FOXP3 Panel

FOXP3 Methylation Panel

Regulatory T-cells (Tregs) play an important role in the immune system by inhibiting self-reactive effector T-cells, maintaining self-tolerance, and thereby preventing autoimmune disease. Tregs are known to exhibit a unique epigenome, and specifically, they express the transcription factor FOXP3, which is a biomarker that distinguishes Tregs from other non-regulatory T-cells. EpigenDx’s FOXP3 panel is a set of validated DNA methylation assays targeting genes that demonstrate the relationship between methylation and Treg differentiation.

The FOXP3 Methylation Assays cover a total of 271 CpG sites, 19 human gene regions, 15 mouse gene regions, 1 rhesus monkey gene region. We can analyze the entire panel using targeted Next-Gen bisulfite sequencing. Or, we can analyze select PCR regions using Pyrosequencing. The panel is customizable to exclude or include any other target regions of your interest.

FOXP3 Panel Available as Kits or Services

Information: Human FOXP3 Methylation Panel
Platform: Targeted Next-Gen Bisulfite Sequencing
Pyrosequencing
Species: Human (Homo sapiens)
Stats: 144 CpG sites over 19 genes and 26 amplicons
Full Gene List: CD274, CD3D_CD3G, CTLA4, ETS1, FOXP3, IKZF2, IKZF4, IL2, IL2RA (CD25), IRF4, ITGAL, MAPRE2, MYC, PD-1 (PDCD1), RUNX1, TNFA, TNFRSF18, TNFRSF25, TOLLIP
Information: Mouse Foxp3 Methylation Panel
Platform: Targeted Next-Gen Bisulfite Sequencing
Pyrosequencing
Species: Mouse (Mus musculus)
Stats: 127 CpG sites over 6 genes and 15 amplicons
Full Gene List: Foxp3, Ctla4, Helios (Ikzf2), Eos (Ikzf4),
Il2ra (Cd25), Tnfrsf18 (Gitr)
EpigenDx FOXP3 Panel Methylation Analysis Service
  • Send your samples to EpigenDx’s service laboratory.
  • Ability to accept all types of DNA (Serum, FFPE, etc.).
  • Versatility with Pyrosequencing and targeted Next-Gen Bisulfite Sequencing
  • EpigenDx provides a complete solution: DNA extraction, bisulfite modification, library preparation, sequencing, and data analysis.
  • Full result reports with an explanation of the data.
FOXP3 Panel Assay Kits
  • Primers compatible with IonTorrent™, all major NGS and Pyrosequencing technologies.
  • Primers good for 300 reactions.
  • Access to our knowledgeable and experienced customer service.

Selected Genes of the FOXP3 Panel

FOXP3

Forkhead-box protein P3 (Foxp3) is a major transcription factor that is necessary for induction of the immunosuppressive function of regulatory T cells (Tregs). It is considered to be the most specific marker for Tregs in tumors, with its expression leading to poor survival rates in some types of cancer and improved survival rates in other types. Studies have shown that epigenetic modifications contribute to the stable expression of FOXP3 and other molecules associated with Treg cell function. These modifications include demethylation of CpG sites in the conserved non-coding sequence 2 (CNS2), Treg specific demethylated region (TSDR), proximal promoter, and upstream enhancer regions of the gene. Understanding the epigenetic regulation of FOXP3 and associated genes is critical in developing new approaches to autoimmune disease and cancer treatment.

Human FOXP3 Methylation Analysis: Product Contents
  • 3 Methylation Assays across the promoter and intron 1 (TSDR and CNS2 region) of Human FOXP3
  • Total coverage of 19 CpG sites

Mouse Foxp3 Methylation Analysis: Product Contents
  • 10 Methylation Assays across the distal promoter, proximal promoter, intron 1 (TSDR and CNS2 region), and 3′ downstream trailer of Mouse Foxp3.
  • Total Coverage of 102 CpG sites

Rhesus Monkey Foxp3 Methylation Analysis: Product Contents
  • 1 Methylation Assays across the homologous TSDR region
  • Total Coverage of 10 CpG sites


CTLA4

CTLA4 is an inhibitory receptor protein found on the surface of FOXP3+ Tregs. The interaction between CTLA4 and receptors on antigen-presenting cells is known to turn off the immune response. While FOXP3 and CTLA4 are usually demethylated and expressed in Tregs, hypermethylation of the CTLA4 promoter has been found to result in dysfunctional FOXP3+ Tregs and autoimmune diseases such as rheumatoid arthritis. Assays covering SNPs in the gene have also been used to study the expression of CTLA4 in T-cells.

Human CTLA4 Methylation Analysis: Product Contents
  • 1 Methylation Assay across intron 1 and exon 2 of Human CTLA4
  • Total coverage of 7 CpG sites

Mouse Ctla4 Methylation Analysis: Product Contents
  • 1 Methylation Assay across intron 1 and exon 2 of Mouse Ctla4
  • Total Coverage of 4 CpG sites



PD-1 (PDCD1)

PD-1, or programmed cell death protein 1, is a receptor on T-cells encoded by the PDCD1 gene that causes T-cell death or inhibition when bound with its ligand PD-L1. High PD-L1 expression has been associated with the reduction of conventional T-cell populations and the induction of FOXP3-expressing Tregs, downregulating the immune response. Thus, PD-1 and PD-L1 inhibitors are useful as cancer immunotherapy treatments.

EpigenDx has designed and validated DNA methylation assays covering the promoter region of PDCD1. Permanent demethylation of the PDCD1 promoter has also been suggested to be a key feature of chronic viral-induced autoimmune diseases such as HIV.

Human PDCD1 Methylation Analysis: Product Contents
  • 2 Methylation Assays across the promoter region of Human PDCD1
  • Total coverage of 20 CpG sites


Helios (IKZF2) and Eos (IKZF4)

Helios (IKZF2) and Eos (IKZF4) are two members of the Ikaros transcription factor family that are demethylated and expressed along with FOXP3 in Tregs. While Eos interacts directly with FOXP3 to silence certain genes (encoding T effector cytokines for instance), Helios binds to the FOXP3 promoter to upregulate FOXP3 expression. Both Eos and Helios are important biomarkers, essential for the proper function of CD4+ FOXP3+ Tregs. EpigenDx has designed and validated DNA methylation assays covering the intronic, non-coding regions of these two genes.

Mouse Ikzf2 Methylation Analysis: Product Contents
  • 1 Methylation Assay across intron 3 of Mouse Ikzf2
  • Total coverage of 6 CpG sites

Mouse Ikzf4 Methylation Analysis: Product Contents
  • 1 Methylation Assay across intron 1 of Mouse Ikzf4
  • Total Coverage of 4 CpG sites


IL2RA (CD25)

Before FOXP3 was discovered as the primary biomarker for the identification of Tregs, early studies used IL2RA (CD25), or the alpha chain of the interleukin-2 receptor, along with CD4 as the primary identifying membrane protein on Treg cells. IL2-IL2R signals play an important role in the maintenance of Tregs in the periphery, allowing suppressive Tregs to respond appropriately to T helper cells secreting IL2. Because naturally arising Tregs constitutively express IL2RA, whereas T helper cells only express IL2 and IL2RA upon activation, IL2RA remains an important biomarker for the epigenetic differentiation of Tregs.

Human IL2RA Methylation Analysis: Product Contents
  • 1 Methylation Assay across intron 1 of Human IL2RA
  • Total coverage of 3 CpG sites

Mouse Il2ra Methylation Analysis: Product Contents
  • 1 Methylation Assay across intron 1 of Mouse Il2ra
  • Total Coverage of 3 CpG sites


IRF4

IRF4 is a transcription factor that physically interacts with FOXP3 to regulate different parts of the Treg genome. The FOXP3-dependent expression of IRF4 is suggested to control the expression of other genes responsible for the suppressive characteristics of Treg cells.

Human IRF4 Methylation Analysis: Product Contents
  • 1 Methylation Assay across the 3′ downstream trailer of Human IRF4
  • Total coverage of 4 CpG sites



TNFRSF18 (GITR)

TNFRSF18, or GITR (glucocorticoid-induced TNF tumor-necrosis factor-related receptor), is another protein that is demethylated and expressed in high levels in Tregs and activated T cells. Because the engagement of GITR with anti-GITR antibodies can inhibit Treg function and deplete Treg population, drugs blocking GITR/TNFRSF18 have been proposed as cancer immunotherapy treatments and allergy medication.

Mouse Tnfrsf18 Methylation Analysis: Product Contents
  • 1 Methylation Assay across exon 4 of Mouse Tnfrsf18
  • Total Coverage of 4 CpG sites